IgG-like bispecific antibodies have different binding specificities on each arm of the antibody. They are similar in structure to monospecific IgGs in that they contain two heavy chains with VH, CH1, CH2 and CH3 regions, and two light chains with VL and CL regions. However, in order to create different specificities on each arm, IgG-like bispecific antibodies preferably have two different heavy chains (at least in the VH regions) that pair in heteromeric or asymmetric fashion as opposed to homodimerically. In addition, assembly of functional bispecific molecules requires complementary pairing of two distinct light chains (different at least in the VL region) to their respective heavy chains.
Various strategies have been proposed to solve the problem of asymmetric heavy chain pairing. One method is to mutate the CH3 domains of the antibodies in order to favor their heterodimerization (i.e. pairing of heavy chain A with heavy chain B) and to prevent their homodimerization. A well known embodiment of this methodology involves the “knob into holes” approach (Ridgeway et al., 1996, Protein Eng. 9(7): 617-621). A “knob” mutation, consisting of the replacement of a small amino-acid by a larger one, is introduced at the CH3 dimer interface of the heavy chain of antibody A, resulting in a steric hindrance which prevents homodimerization. Concurrently in order to promote heterodimerization, a complementary “hole” mutation, consisting of the replacement of a large amino acid by a smaller one, is introduced into the CH3 domain of antibody B.
Another method is the use of electrostatics, wherein the CH3 domain interface of the antibody Fc region is modified with selected mutations to create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (see e.g., Gunasekaran et al., 2010, J Biol Chem. 285(25): 19637-19646, WO/2006/106905, U.S. Patent Application 2011/0123532 each of which is incorporated herein by reference). Yet other methods for asymmetric heavy chain pairing have also been published (see Merchant et al., 1998, Nat Biotechnol 16: 677-681; Moore et al., 2011, MAbs 3:546-567; and Davis J H et al., 2010, Protein Eng Des Sel 23: 195-202 and U.S. Patent Application 2012/0149876 each which are accomplished herein by reference).
Quadroma technology has also been used to create bispecific antibodies. Quadroma technology (Milstein and Cuello, 1983, Nature, 305(5934): p. 537-40) is based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. But because of the random pairing of two different Ig heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different immunoglobulin species are generated of which only one is the functional bispecific antibody. The presence of heavy chain/light chain mispaired by-products and significantly reduced production yields means that sophisticated purification procedures are required to obtain functional bispecific antibodies in sufficient quantities.
In an attempt to reduce heavy chain/light chain mispairing, methods were employed using antibodies having different specificities but sharing a common light chain, previously identified from an scFv phage library (see e.g., Merchant et al., 1998, Nat Biotechnol 16: 677-681; U.S. Pat. No. 7,183,076). The drawback of this approach is the difficulty in identifying antibodies having a common light chain. That is, while it is possible for antibodies to be engineered to bind antigen with negligible energetic contribution of the light chain thus allowing for a common light chain to be used when engineering a bispecific IgG, it is more common for antibodies to require energetic contributions of both the heavy chain and light chain for high affinity binding to antigen. When two monospecific antibodies are reformatted as a single bispecific antibody, the correct pairing of each heavy chain with their corresponding light chain becomes important for the retention of binding properties of the original monospecific IgG antibodies. This significantly limits the usage of common light chains when reformatting existing antibodies as bispecifics. Without additional molecular mechanisms to ensure correct heavy/light chain pairing, coexpression of the two heavy chains and two light chains would result in mixed products with mispaired heavy and light chains.
Despite such attempts there is a need in the art for improved methods of producing multispecific antibodies, including bispecific antibodies, that exhibit relatively high fidelity in pairing heavy and light chains and provide assembled constructs that have high affinity for both antigens.